RAG2 is a member of the rice 14-16 kDa α-amylase/trypsin inhibitors, which are the main allergens in rice grains. Recombinant RAG2 protein was expressed in Escherichia coli, and polyclonal antisera were prepared against the protein. The polyclonal antisera specifically bound with 14-16 kDa α-amylase/ trypsin inhibitors from husked brown, red and black rice. No cross-reaction was observed between the polyclonal antisera and saline-soluble proteins from other tested cereal grains. By immunoblotting with the polyclonal antisera, 14-16 kDa α-amylase/trypsin inhibitors were found to be distributed throughout the endosperm of brown rice and their contents were estimated. Moreover, 14-16 kDa α-amylase/trypsin inhibitors were also detected in processed foods. Thus, the polyclonal antisera prepared against recombinant RAG2 protein could be used to compare the content of 14-16 kDa α-amylase/trypsin inhibitors in rice samples and probe the presence of the allergen in processed foods.Keywords: rice (Oryza sativa), RAG2, α-amylase/trypsin inhibitor, recombinant protein, polyclonal antisera, processed food *To whom correspondence should be addressed. E-mail: kaz@affrc.go.jp IntroductionCereal allergy is of great concern in Japan, as it is in other countries where cereals are a staple food and the major nutrition source. Rice is the second most important cereal crop in the world, after wheat, and is consumed by more than half of the world's population. The association between rice and allergic reactions was first reported in Japanese patients with a history of asthma induced by exposure to rice flour and eczema exacerbated by rice ingestion (Shibasaki et al., 1979). Rice-induced allergy is IgE-mediated in the case of bronchial asthma (Arai et al., 1998;González-Mendiola et al., 2003;Lezaun et al., 1994) or atopic dermatitis (Di Lernia et al., 1992;Ikezawa et al., 1992) associated with the ingestion of cooked rice or inhalation of vapor during its cooking. Although rice is commonly thought of as a "hypoallergenic" food, the clinical importance of rice allergy has been demonstrated by a report in which a high radioallergosorbent test (RAST) score for rice allergens had a close relationship to the development of severe atopic dermatitis (Ikezawa et al., 1992). Moreover, Yamada et al. (1985) reported that rice has apparently exceeded other cereals in allergenicity, and ranked second after egg whites as the most common potential allergy-causing food, as assessed by an IgE RAST. A recent report (Mehr et al., 2009) noted that rice was a common trigger of a severe type of allergic reaction called food proteininduced enterocolitis syndrome (FPIES) in children.Several rice proteins with molecular masses of 14-16, 26, 33 and 60 kDa were demonstrated to be recognized by serum IgE of patients showing hypersensitive reactions to rice ingestion (Urisu et al., 1991;Usui et al., 2001). The first reported allergen in rice was a 16 kDa protein with reactivity for IgE of rice-hypersensitive patients (Matsuda et al., 1991). The 16 kDa protein is ...

We aimed to detect the allergen proteins in food materials using recently developed near-infrared fluorescent probes. Sensitivities of this method were comparable to chemiluminescence detection methods, which are known to be sensitive. In addition, the sensitivities of this near-infrared fluorescent method were at least +*ῌ/*-times higher than those of the conventional visible fluorescent methods using Cy-and Cy/ dyes. This method was e#ectively applicable to immunoblotting, dot-blotting and plate-assay (direct FLISA : fluorescence-linked immunosorbent assay) with ELISA plate. Allergen levels of the food sample were quantified by standard curves using standard allergen protein using the dot-blotting technique. This highly sensitive detection system also provided multiple detections of di#erent allergens for di#erent antibodies and dyes with distinct properties of wavelength. This enables high-throughput screening of characteristic allergen contents of target food materials, or cultivars. Generally, allergen proteins are recognized by patient's serum IgE. Therefore, we tried to detect patient's IgE-binding proteins, the putative allergens in foodstu#s. In this detection system, it was possible to detect IgE-binding proteins with sensitivity almost equivalent to a chemiluminescent detection system. Taken together, it was shown that this novel detection system was an e#ective technique for the sensitive detection and screening of food allergens.

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